The present invention relates to a novel interferon-xcex1 (hereinafter referred to as IFN-xcex1). More preferably, the present invention relates to a novel human IFN-xcex1 and its derivative having an unprecedentedly high specific activity, and a gene thereof, as well as medical uses of said IFN-xcex1 and its derivative.
IFN is a generic term for proteins having anti-viral activity, among which those produced from leukocytes or lymphoblastic cells by stimulation with virus or double stranded nucleic acids are termed as IFN-xcex1. IFN-xcex1 has a variety of activities including anti-viral activity and a cellular growth-suppressing activity, which activities have been found to be useful in a variety of diseases such as hepatitis type B, hepatitis type C, and cancer.
Analysis of base sequences of IFN-xcex1 genes cloned from a variety of DNA libraries have revealed that IFN-xcex1 has several subtypes (Science 209: 1343-7 (1980), Gene 11: 181-6 (1980), Nature 290: 20-26 (1981), Nature 313: 698-700 (1985), J. Invest. Dermatol. 83: 128s-136s (1984)). For example, for the main subtype gene of IFN-xcex12, three types(xcex12a, xcex12b, and xcex12c) have been identified (J. Interferon Res. 2: 575-85 (1982), J. Interferon Res. 13: 227-31 (1993), J. Biol. Chem. 268: 12565-9 (1993), Acta Virol. 38: 101-4 (1994), Biochim. Biophys. Acta. 1264: 363-8 (1995)). In addition, there are currently known nearly 20 types of subtype genes including IFN-xcex11a, -xcex11b, -xcex14a, -xcex14b, -xcex15, -xcex16, etc.
On the other hand, vigorous efforts have been made in structural analysis of proteins in stead of genes, that is to purify each subtype of natural IFN-xcex1 and then to analyze its primary structure. A group in Wellcome, for example, made an attempt on structural analysis using a mixture of two fractions separated by gel filtration of purified IFN derived from Namalwa cells, human lymphoblastic cells, and, as a result, have demonstrated the structure, though not complete, of IFN-xcex11 and IFN-xcex12 (Nature 287: 408-11 (1980)). As a result of intensive efforts to purify Namalwa cell-derived IFN subtypes , Zoon et al. of FDA have successfully isolated several subtypes and revealed their partial structure, anti-viral activity, cellular growth-suppressing activity, and NK cell-inducing activity (Infect. Immun. 34: 1068-70 (1981), J. Biol. Chem. 267: 15210-6 (1992), J. Biol. Chem. 268: 12591-5 (1993)). Furthermore, in the analysis of the primary structure for one major subtype, they have demonstrated that it was IFN-xcex12b (J. Biol. Chem. 267: 15210-6 (1992)).
As stated above, IFN-xcex1 has various subtypes, of which base sequences and amino acid sequences are being elucidated, though the structure and physical properties of all subtypes have not been revealed.
The present invention intends to provide a novel IFN-xcex1 and its gene. Thus, the present invention intends to provide a novel human IFN-xcex1, its derivative having an unprecedentedly high specific activity, a gene encoding them, and a pharmaceutical agent comprising said IFN-xcex1 and its derivative as active ingredient.
The inventors of the present invention have attempted to isolate major subtypes contained in IFN-xcex1 derived from human natural-type lymphoblastic cells (hereinafter referred to as HLBI). Thus, the inventors have found that the subtypes can be easily separated by means of a reverse-phase HPLC that utilizes xcexcBondasphere column and Vydac(trademark)-C4 column and thereby have successfully isolated and purified 12 major subtypes contained in HLBI.
From the analysis of the N-terminal amino acid sequence and the primary structure of the isolated subtypes, it was found that a novel IFN-xcex1 subtype was contained in addition to the existing IFN-xcex11, xcex12b, xcex15, xcex17, xcex18, xcex114, xcex117 and xcex121. The inventors of the present invention have termed this novel IFN-xcex1 subtype as IIIe.
On these subtypes, anti-viral activity against Sindbis virus was determined using human-derived cultured cells, FL cells, and it was found that the anti-viral activity of a major subtype IFN-xcex12b was 1.67xc3x97108 u/mg whereas the novel IFN-xcex1 subtype IIIe had the highest and unprecedentedly high specific activity of 4.3-5.2xc3x97108 u/mg.
Furthermore, the identification of the entire amino acid sequence of the subtype IIIe revealed that the primary structure of the subtype IIIe was similar to an amino acid sequence deduced from the sequence of IFN-xcex110a (=xe2x88x92xcex1C) gene as reported in Nature Mar. 5, 1981; 290, 20-26, but had a novel amino acid sequence in which the amino acid at position 19 was Ala in stead of Gly. The cloning of said IIIe gene also revealed that it is different from IFN-xcex110a by three bases on the base sequence level.
As described above, the IFN-xcex1 subtype IIIe of the present invention has an unprecedentedly high specific activity, and thereby its dosage can possibly be reduced compared to commercially available recombinant human IFN-xcex12a, recombinant human IFN-xcex12b, etc. Furthermore, it is expected to exhibit effectiveness on cases with HCV-Genotype II, high virus level etc. on which conventional IFN is believed to be not very effective.
The present invention was completed based on the above findings.
Thus, the present invention relates to the following (1) to (13):
(1) DNA comprising the base sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or DNA encoding a protein comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
(2) DNA encoding a derivative of human interferon-xcex1, said DNA being selected from
(A) DNA hybridizing to the DNA according to the above (1) under a stringent condition, and
(B) DNA encoding a protein in which one or a plurality of amino acid residues of a protein encoded by the DNA according to the above (1) have been replaced, deleted, and/or added,
wherein the protein encoded by said DNA has the following characteristics (a) and (b):
(a) having a specific activity higher than 4.0xc3x97108 units/mg as measured by an anti-viral activity assay on Sindbis virus using the FL cell, a human-derived cultured cell; and
(b) migrating as a band with an apparent molecular weight of 20 kDa-23 kDa on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction treatment;
(3) The DNA according to the above (2) which encodes a protein comprising an amino acid sequence in which 1-5 amino acid residues in the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4 have been replaced, deleted, and/or added;
(4) An expression vector having the DNA according to any one of the above (1)-(3);
(5) A transformant transformed with the expression vector according to the above (4);
(6) A method of producing a recombinant human interferon-xcex1 or its derivative, which method comprises culturing the transformant according to the above (5) and recovering the expressed recombinant human interferon-xcex1 or its derivative;
(7) A human interferon-xcex1 or its derivative which is encoded by the DNA according to any one of the above (1)-(3) or produced by the production method according to the above (6);
(8) A human interferon-xcex1 comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4;
(9) A human interferon-xcex1 or its derivative according to the above (7) or (8) or a pharmaceutically acceptable salt thereof for use as active ingredient of a pharmaceutical composition;
(10) A pharmaceutical composition comprising the human interferon-xcex1 or its derivative according to the above (7) or (8) or a pharmaceutically acceptable salt thereof as active ingredient together with a pharmaceutically acceptable carrier or excipient;
(11) The pharmaceutical composition according to the above (10) which is for treatment of viral diseases;
(12) The pharmaceutical composition according to the above (10) which is for treatment of cancer;
(13) A method of treating viral diseases or cancer which method comprises administering to a mammal including a human an effective amount of the human interferon-xcex1 or its derivative according to the above (7) or (8) or a pharmaceutically acceptable salt thereof.
The DNA of the present invention encodes a novel human IFN-xcex1 and its derivative, and specifically there can be mentioned DNA comprising the base sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or DNA encoding a protein comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
Also encompassed in the scope of the present invention is DNA that hybridizes to the DNA described above under a stringent condition, or DNA encoding a derivative of a human interferon-xcex1 selected from DNA encoding a protein in which one or a plurality of amino acid residues of the protein encoded by the above DNA have been replaced, deleted, and/or added, wherein the protein encoded by said DNA has the characteristics of (a) having a specific activity higher than 4.0xc3x97108 units/mg as measured by an anti-viral activity assay on Sindbis virus using the FL cell, a human-derived cultured cell, and (b) migrating as a band with an apparent molecular weight of 20 kDa-23 kDa on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction treatment. The DNA of the present invention will now be sequentially explained below.
1) DNA Encoding the IFN-xcex1 Subtype IIIe
Among the above DNA, xe2x80x9cDNA comprising the base sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2xe2x80x9d and xe2x80x9cDNA encoding a protein comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4xe2x80x9d are DNA encoding the human-derived IFN-xcex1 subtype IIIe of the present invention. Among them, the base sequence as set forth in SEQ ID NO: 1 and the amino acid sequence as set forth in SEQ ID NO: 3 are a base sequence and an amino acid sequence corresponding to the full-length subtype IIIe including the signal peptide, and the base sequence as set forth in SEQ ID NO: 2 and the amino acid sequence as set forth in SEQ ID NO: 4 are a base sequence and an amino acid sequence corresponding to the full-length mature type subtype IIIe including no signal peptide.
Said DNA can be cloned by the PCR method described in Example 3 below. As templates for performing PCR, genomic DNA or cDNA derived, for example, from the Namalwa cell (ATCC(trademark) No. CRL-1432 etc.) may be used, and as primers, a primer comprising the base sequence, for example, as set forth in SEQ ID NO: 6 and SEQ ID NO: 7 may be mentioned.
Furthermore, cloning may also be performed by modifying amino acids based on the known IFN-xcex1 subtypes reported in Nature 290: 20-26 (1981), etc. Said cloning may be readily performed by a person skilled in the art according to Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989) etc.
2) DNA Encoding a Derivative of IFN-xcex1 Subtype IIIe
Among the above DNA, xe2x80x9cDNA that hybridizes to the DNA of the subtype IIIe under a stringent conditionxe2x80x9d and xe2x80x9cDNA encoding a protein in which one or a plurality of amino acid residues of the amino acid sequence of the subtype IIIe have been replaced, deleted, and/or addedxe2x80x9d mean DNA encoding a protein having a structure similar to the subtype IIIe such as an artificially constructed, so-called modified protein, an allele mutant present in the living body, and an IFN-xcex1 subtype similar to IIIe. Hereinbelow, protein having such a structure similar to the subtype IIIe will be termed as a xe2x80x9cderivative.xe2x80x9d
As used herein, as a method of producing xe2x80x9cDNA encoding protein in which one or a plurality of amino acid residues of the amino acid sequence of the subtype IIIe have been replaced, deleted, and/or added,xe2x80x9d there can be known methods such as site-directed mutagenesis and the PCR method, which may be easily performed by a person skilled in the art according to Nucleic Acid Res. 10: 6487 (1982), Methods in Enzymology 100: 448 (1983), Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach, IRL Press, pp. 200 (1991), etc.
As the number of amino acid residues to be modified, there can be mentioned those numbers that may be replaced, deleted, and/or added by known methods such as the site-directed mutagenesis mentioned above. Since IFN-xcex1 is a relatively small protein of which mature type comprises 166 amino acids, the number of amino acid residues to be modified is preferably 10 or less, and more preferably 5 or less. For sites that are important for activity expression, modification is preferably substitution to conservative amino acids.
As used herein, as a method of producing xe2x80x9cDNA that hybridizes to the DNA of the subtype IIIe under a stringent condition,xe2x80x9d there can be mentioned known methods such as a PCR method, and a hybridization method. Specifically, it may be performed according to the method described in the above Molecular Cloning.
As used herein, xe2x80x9cunder a stringent conditionxe2x80x9d means a condition in which hybridization is performed at 42xc2x0 C. in a solution containing 6xc3x97SSC (20xc3x97SSC represents 333 mM sodium citrate and 333 mM NaCl), 0.5% SDS, and 50% formamide, followed by washing at 68xc2x0 C. in a solution of 0.1xc3x97SSC and 0.5% SDS, a condition as described in the above-mentioned Molecular Cloning, or the like. More preferably, there can be mentioned a condition in which hybridization occurs only for those that are different from the DNA of the subtype IIIe by about 1-5 amino acids.
Among the above DNA, the DNA encoding a protein which has the following characteristics can be the DNA of the present invention:(a) having a specific activity higher than 4.0xc3x97108 units/mg as measured by an anti-viral activity assay on Sindbis virus using human-derived cultured cells, FL cells; and (b) migrating as a band with an apparent molecular weight of 20 kDa-23 kDa on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction treatment.
Whether the protein encoded by the candidate DNA that can be the DNA of the present invention satisfies the above requirement (a) can be evaluated by performing an anti-viral activity assay as described below.
Thus, 45,000-60,000 FL cells (The National Institute of Health, ATCC(trademark) etc.) prepared in a 10 v/v % bovine calf serum-Eagle""s minimum essential medium are inoculated into each well of a microtiter plate, which is incubated in a 5% carbon dioxide incubator at 37xc2x0 C. for 20 hours. Then 100 xcexcl of the candidate IFN sample is added to each well and incubated at 37xc2x0 C. for 6 hours. The culture liquid is discarded and 105-106 PFU of Sindbis virus (The National Institute of Health, ATTC etc.) per well is added, and incubated at 37xc2x0 C. for 2 days. The cells are stained in a 0.02 w/v % Neutral red-5 v/v % bovine calf serum-Eagle""s minimum essential medium, and the degree of cytopathic effect is determined by the amount of the dye incorporated.
As methods of calculating titer, the following method may be mentioned. Thus, the dye incorporated into the cell is eluted with an acidified 30 v/v % ethanol and absorbance is determined at a wavelength of 545 mxcexc. The experimental titer of the sample and the standard (The National Institute of Health) are calculated from the dilution factor of the sample exhibiting 50% of the absorbance of the dye incorporated into the normal cell and that of the standard. The titer of the standards and the experimental titer are used to determine a correction factor, which is used to correct the experimental titers of the samples to obtain the titers of the samples. In the above activity assay, those having a specific activity higher than 4.0xc3x97108 units/mg are included in the scope of the present invention.
Whether the protein encoded by the candidate DNA satisfies the above requirement (b) can be detected by subjecting the candidate protein to reduction treatment with 2-mercaptoethanol followed by a normal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then staining with Coomassie brilliant blue. The density of the SDS-PAGE gel at this time is preferably about 3.5% for the concentration gel and about 15% for the separation gel. Electrophoresis is preferably performed at about 50 mA. In the SDS-PAGE as described above, those having an apparent molecular weight of 20 kDA-23 kDa are included in the scope of the present invention.
By recombinant DNA technology using the DNA of the present invention, the protein of the present invention, that is, the novel human IFN-xcex1 subtype IIIe and its derivative can be produced in large quantities. In order to produce the recombinant human IFN-xcex1 and its derivative of the present invention by expressing the DNA of the present invention, methods are used, for example, based on many textbooks and references including the above Molecular Cloning. For human IFN-xcex12a and -xcex12b, their recombinant types have already been produced and are commercially available. Based on the production method for known IFN-xcex1, the novel human IFN-xcex1 and its derivative of the present invention can be produced in large quantities (see Japanese Examined Patent Publication (Kokoku) No. 63-63198, Japanese Examined Patent Publication (Kokoku) No. 3-21151, Nucleic Acids Res. 8: 4057 (1981), Nature 287: 411 (1980), Proc. Natl. Acad. Sci. USA 77: 5230 (1980), and the like).
Specifically, by optionally adding a regulatory gene such as a promoter sequence (for example, trp, lac, T7, and SV40 early promoter) that controls transcription to the upstream of the DNA to be expressed, which is then integrated into a suitable vector (for example, PBK-CMV, pCAGGS, and pZeoSV), it is possible to construct an expression vector that is replicated and expressed in the host cell. Then said expression vector is introduced into a suitable host cell to obtain a transformant. As the host cell, there can be mentioned a prokaryote such as Escherichia coli, a unicellular eukaryote such as yeast, a multicellular eukaryote such as an insect or an animal, and the like. As the method of introducing an expression vector into a host cell, a known method can be used such as the calcium phosphate method, the DEAE-dextran method, and the electric pulse method. By culturing the thus obtained transformant in a culture medium suitable for said transformant by a standard method, the desired recombinant human IFN-xcex1 and its derivative of the present invention can be produced. The recombinant human IFN-xcex1 and its derivative of the present invention obtained in this manner can be isolated and purified by a common biochemical method using, for example, anti-IFN-xcex1 antibody.
Furthermore, they can also be obtained by purifying a subtype obtained by using as raw material the human natural type lymphoblast-derived IFN-xcex1 (HLBI) (manufactured by Sumitomo Pharmaceutical Co., Ltd.) as described in Example 1 below.
The novel human IFN-xcex1 subtype IIIe and its derivative of the present invention thus obtained are encoded by the above DNA of the present invention, and they are proteins produced by the expression of the latter. As a specific example, there may be illustrated the novel human IFN-xcex1 subtype IIIe of the present invention comprising the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
The novel IFN-xcex1 and its derivative of the present invention can be used as active ingredient of pharmaceutical agents. Thus the present invention also intends to provide a pharmaceutical composition comprising a novel IFN-xcex1 and its derivative or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or excipient, and optionally with another therapeutic and/or preventive agent.
It is conventionally known that IFN-xcex1 has a variety of effects including an anti-viral effect, a cellular growth-suppressing effect, a natural killer cell-activating effect, and the like. Accordingly, the novel IFN-xcex1 of the present invention is also expected to be able to treat various diseases based on these effects.
As the indicated diseases, cancer (malignant tumor), viral diseases, and immunological diseases may be mentioned, and specifically there can be mentioned kidney cancer, renal-cell carcinoma, breast cancer, bladder cancer, basal cell carcinoma, head and neck cancer, cervical dysplasia, skin carcinoma, Kaposi""s sarcoma, malignant melanoma, non-Hodgkin lymphoma, infant hemangioma, chronic granulomatosis, type B chronic hepatitis, type C chronic hepatitis (active, non-active), herpes infections (genital herpes, corneal herpes inflammation, oral herpes inflammation, etc.), chronic myelocytic leukemia (CML), adult T cell leukemia, hairy cell leukemia, hairy cell leukemia, T cell leukemia virus (HTLV-1) myelopathy, multiple myeloma, lymphoma, subacute sclerosing panencephalitis (SSPE), Sjogren""s syndrome, condyloma acuminata, AIDS, multiple sclerosis (MS), stomatitis, genital wart, intravaginal wart, erythrocytosis, thrombocythemia, psoriasis, mycosis fungoides, sudden deafness, senile disciform macular degeneration, Paget""s disease, and the like.
Since the IFN-xcex1 and its derivative of the present invention has a specific activity higher than the conventional ones, they are expected to be effective on cases with HCV-Genotype II, high virus level etc. on which conventional IFN is said to be not very effective.
The novel interferon-xcex1 and its derivative of the present invention or a pharmaceutically acceptable salt thereof may be administered as a pharmaceutical composition via an oral or parenteral (for example, intravenous, subcutaneous or intramuscular injection, local, transrectal, transdermal, or nasal) route. As compositions for oral administration, there can be mentioned, for example, tablets, capsules, pills, granules, powders, liquids, and suspensions, and as compositions for parenteral administration, there can be mentioned, for example, aqueous or oily agents for injection, ointments, creams, lotions, aerosols, suppositories, and adhesives. It is also possible to prepare sustained release minipellet formulations and implant them near the affected area, or to gradually administer them to the affected area on a continuous basis using an osmotic pump. These formulations may be prepared using conventionally known technology, and may contain non-toxic and inert carriers or excipients that are commonly used in the field of pharmaceutics.
The above pharmaceutical compositions may be manufactured by blending the active ingredient of the present invention with pharmaceutically acceptable conventional carriers, excipients, binders, stabilizers, buffers, solution adjuvants, or tonicity agents. When used as injections, there may be added buffers, solution adjuvants, tonicity agents etc.
The dosage and the frequency of administration may vary depending on the condition and history of the disease, age and weight of the patient, dosage form, etc., but when they are administered to adults (body weight 60 kg) via a parenteral (for example, intravenous) route, they are generally prepared, as appropriate, in the range of 0.001-1 mg per day, preferably 0.005-0.5 mg, and most preferably 0.010-0.2 mg, and administered in single or several divided doses.